Extended Data Fig. 5: P2RY8 expression and distribution in human tonsil.
From: S-Geranylgeranyl-l-glutathione is a ligand for human B cell-confinement receptor P2RY8
a, qPCR for expression of P2RY8 in the indicated subsets sorted from human tonsil, relative to PTPRC. (n = 3) b, Immunofluorescence for P2RY8 (green) and CD4 (red) in PFA-fixed human tonsil sections. Inset depicts P2RY8+ and P2RY8high expressing CD4+ cells within the germinal centre and at the germinal-centre border. Scale bars, 50 µm. c, Intracellular flow cytometry using the anti-P2RY8 antibody from b, which binds the C terminus of P2RY8, on empty vector-GFP- or P2RY8-GFP-transduced WEHI-231 (mouse) cells, compared with rabbit isotype control or no primary antibody staining conditions. d, Intracellular flow cytometry for P2RY8 in tonsil IgD+CD38− follicular B cells, IgD−CD38+ germinal-centre B cells, CXCR5−CD4+ T cells or CXCR5+PD-1+ TFH cells. e, Intracellular flow cytometry for P2RY8 in Ly8 cells edited using CRISPR–Cas9 with a control non-targeting guide (red) or a guide targeting P2RY8 (black). f, TIDE analysis of edited Ly8 cells showing editing efficiency around the expected cut site. g, pAKT levels in DOHH2 cells transduced with either GNA13 or empty vector, treated as in Fig. 3a (n = 5). h, pAKT levels in P2RY8-expressing or control WEHI-231 cells, treated as indicated (n = 9). Data are representative of or pooled from three (a), four (b) or two (d) tonsils; and four (h), two (c, e, g) or one (f) experiments. Graphs depict mean with s.d. Points represent biological replicates. P values determined by one-way ANOVA with Bonferroni’s multiple comparisons test (g, h).
